The goals of this proposal is to provide a detailed understanding of the activities of five proteins from Escherichia coli that are involved in recombinational DNA repair and homologous genetic recombination. These are the RuvA, RuvB, RecF, RecO, and RecR proteins. Some mutations in each of the corresponding genes lead to recombination defects in certain genetic backgrounds. The same mutations confer sensitivity to DNA damaging agents in an otherwise wild type background. The RecF, O, and R proteins are believed to act early in recombinational processes. One function identified to date is to facilitate RecA protein binding to SSB-coated single-stranded DNA. The amino acid sequence of each of these proteins features a consensus nucleotide binding site, although NTP hydrolysis has not been reported and little is known about the function of nucleotides in any activity these proteins may have. We propose to characterize the DNA and NTP binding properties of the RecF, O, and R proteins in detail. We will also examine the effects of these proteins on RecA activities in vitro. If RecA forms mixed filaments with these proteins or any subset of them, we will focus on defining the activities of the filaments so modified. The RuvA and RuvB proteins are believed to contribute to the processing of branched recombination intermediates generated by RecA protein. These proteins promote DNA branch migration in vitro which could contribute to the rapid generation of hybrid DNA during recombination. The proteins exhibit a limited DNA helicase activity, and RuvB protein hydrolyzes ATP. In combination with RecA, they promote the bypass of regions of heterology that are too long to be bypassed by RecA protein alone. If a barrier cannot be bypassed, these proteins promote an efficient reversal of RecA- mediated DNA strand exchange. One function of these proteins may be to act as a kind of anti-recombination system, to process or reverse stalled Holliday structures that may result from potentially deleterious intragenomic recombination events. Our studies on RuvA and B will focus in part on the characterization of DNA binding and ATP hydrolysis to elucidate the mechanism by which these proteins translocate on the DNA. We will also examine the mechanism by which RuvA and B process stalled DNA branches generate by RecA, as a model for RuvA and B action in all recombination processes.